The objective of the proposed studies is to identify interactions responsible for selective metabolism by cytochrome P450 2A13, a human lung-specific enzyme. 2A13 has the highest known activity for activation of 4-(methylnitrosamino)-1-(3-pyridy!)-1-butanone (NNK), a primary carcinogen in tobacco. In vivo decreases in 2A13 activity have been correlated with substantial reductions in human lung adenocarcinoma, suggesting selective inhibition of 2A13 as a potential chemoprevention strategy. Design of selective inhibitors requires structural information that is lacking for P450s involved in procarcinogen activation, including 2A13. Earlier studies on the structure and function of drug-metabolizing hepatic P450s have identified a general set of active site residues whose molecular characteristics define substrate specificity. The central hypothesis of this proposal is that specific interactions between one or more of the corresponding 2A13 active site residues and preferred 2A13 substrates precisely dictate metabolism by 2A13. This hypothesis will be tested by a combination of experimental approaches, including site-directed mutagenesis, heterologous expression in E. coli, a variety of functional assays and enzyme inhibition studies, and X-ray crystallography. The individual specific aims are to: 1) determine X-ray crystal structures of 2A13, 2) define the contributions of individual active site and naturally polymorphic residues to 2A13 protein structure and function, and 3) evaluate the selectivity of known and suspected family 2A inhibitors for 2A13 versus 2A6. The research proposed in this application is significant because it is expected to generate new and important information on the specific interactions of extrahepatic cytochromes P450 with their ligands. These results may provide an improved scientific basis for understanding the differential substrate selectivity of related P450s in lung and liver and the molecular results of procarcinogen activation. The ultimate goal is to develop and evaluate chemoprevention strategies via 2A13 inhibition. The proposed research is expected to elucidate structural and functional differences between a lung enzyme that activates tobacco carcinogens and a closely-related liver enzyme that removes foreign chemicals from the body. These differences could be exploited to understand differences in cancer risk between individuals and to develop methods to prevent tobacco-associated lung cancer.